The Cardiac Cycle
The term “cardiac cycle” is used to refer to all or any of the mechanical events related to the coronary blood flow or blood pressure that occurs from the beginning of one heartbeat to the beginning of the next. Blood pressure increases and decreases throughout the cardiac cycle. The frequency of the cardiac cycle is the heart rate. Every single ‘beat’ of the heart involves five major stages: (1)“late diastole,” which is when the semilunar valves close, the atrioventricular (Av) valves open and the whole heart is relaxed; (2) “atrial systole,” which is when the myocardium of the left and right atria are contracting, AV valves open and blood flows from atrium to ventricle; (3) “isovolumic ventricular contraction,” which is when the ventricles begin to contract, AV and semilunar valves close, and there is no change in volume; (4) “ventricular ejection,” which is when the ventricles are empty but still contracting and the semilunar valves are open; and (5) “isovolumic ventricular relaxation,” when pressure decreases, no blood is entering the ventricles, the ventricles stop contracting and begin to relax, and the semilunar valves are shut because blood in the aorta is pushing them shut. The cardiac cycle is coordinated by a series of electrical impulses that are produced by specialized heart cells found within the sino-atrial node and the atrioventricular node.
Coronary Blood Flow
The flow of blood through the coronary arteries is pulsatile, with characteristic phasic systolic and diastolic flow components. Systolic flow, which relates to the contraction or pumping phase of the heart cycle, has rapid, brief, retrograde responses. Diastolic flow, which relates to the relaxation or filling phase of the heart cycle, occurs during the relaxation phase after myocardial contraction, with an abrupt increase above systolic levels and a gradual decline parallel with that of aortic diastolic pressures. Intramural coronary blood volume changes during each heartbeat, with the myocardium accommodating the volume change brought about by muscular contraction. Coronary venous flow is out of phase with coronary arterial flow, occurring predominantly in systole and nearly absent during diastole.
For each heartbeat, blood pressure varies between systolic and diastolic pressures. The term “systolic pressure” refers to the peak pressure in the arteries, which occurs near the end of the cardiac cycle when the ventricles are contracting. The term “diastolic pressure” refers to the minimum pressure in the arteries, which occurs near the beginning of the cardiac cycle when the ventricles are filled with blood.
Coronary blood flow not only is phasic but also varies with the type of vessel and location in the myocardium. Coronary arterioles appear to have specialized regulatory elements along their length that operate “in series” in an integrated manner. A system of multiple functional “valves” permits fine control of the coronary circulation. The smallest arterioles dilate during metabolic stress, resulting in reduced microvascular resistance and increased myocardial perfusion. Stenosis or narrowing of a blood vessel produces resistance to blood flow related directly to the morphologic features of the stenosis. As the upstream arteriolar pressure decreases due to a fall in distending pressure across the stenosis, myogenic dilation of slightly larger arterioles upstream occurs and causes an additional decrease in resistance. Increased flow in the largest arterioles augments shear stress and triggers flow-mediated dilation, further reducing the resistance of this network.
The arterial and venous pulsatile flow characteristics of the heart are dependent on intramyocardial compliance. The term “compliance” refers to a measure of the tendency of a hollow organ to resist recoil toward its original dimensions upon removal of a distending or compressing force. The higher the compliance the more elastic the material. Compliance is calculated using the following equation, where ΔV is the change in volume, and ΔP is the change in pressure:C=ΔV/ΔP 
The capacity of the heart as a reservoir is controlled by resistance arterioles to coronary vascular inflow. Outlet resistance is related to intramural cardiac veins. The intramyocardial capillary resistance influences both arterial and venous responses but predominantly acts in concert with outlet resistance.
Approximately 75% of total coronary resistance occurs in the arterial system, which comprises conductance (R1), prearteriolar (R2) and arteriolar and intramyocardial capillary vessels (R3). Normal epicardial coronary arteries in humans typically are 0.3 to 5 mm in diameter, and do not offer appreciable resistance to blood flow. Normally, large epicardial vessel resistance (R1) is trivial until atherosclerotic obstructions compromise the lumen. Precapillary arterioles (R2), 100 to 500 μm in size) are resistive vessels connecting epicardial to myocardial capillaries and are the principal controllers of coronary blood flow. They contribute approximately 25% to 35% of total coronary resistance. Distal precapillary arteriolar vessels (<100 μm in diameter), the main site of metabolic regulation of coronary blood flow, are responsible for 40-50% of coronary flow resistance. The dense network of about 4000 capillaries per square millimeter ensures that each myocyte is adjacent to a capillary. Capillaries are not uniformly patent (meaning open; affording free passage), because precapillary sphincters regulate flow according to the needs of the myocardium.
Several conditions, such as left ventricular hypertrophy, myocardial ischemia, or diabetes, can impair the microcirculatory resistance (R3), blunting the maximal absolute increase in coronary flow in times of increased oxygen demand.
Ischemia
The myocardium depends almost entirely on aerobic metabolism, since oxygen stores in the heart are meager. Myocardial oxygen supply rises and falls in response to the oxygen (energy) demands of the myocardium. The term “autoregulation” refers to the ability to maintain myocardial perfusion at constant levels in the face of changing driving forces. Autoregulation maintains coronary perfusion at relatively constant levels over a wide range of mean aortic pressure. When aortic pressure exceeds its upper or lower limits, coronary blood flow precipitously declines or increases proportionately.
The heart needs to be supplied with a sufficient quantity of oxygen to prevent underperfusion. When reduced perfusion pressure distal to stenoses is not compensated by autoregulatory dilation of the resistance vessels, ischemia, meaning a lack of blood supply and oxygen, occurs. Because the zone least supplied generally is the farthest out, ischemia generally appears in areas farthest away from the blood supply.
After total or near-total occlusion of a coronary artery, myocardial perfusion occurs by way of collaterals, meaning vascular channels that interconnect epicardial arteries. Collateral channels may form acutely or may preexist in an under-developed state before the appearance of coronary artery disease. Preexisting collaterals are thin-walled structures ranging in diameter from 20 μm to 200 μm, with a variable density among different species. Preexisting collaterals normally are closed and nonfunctional, because no pressure gradient exists to drive flow between the arteries they connect. After coronary occlusion, the distal pressure drops precipitously and preexisting collaterals open virtually instantly.
The term “myocardial ischemia” refers to a decrease in blood supply and oxygen to the cells of the myocardium. The development of myocardial ischemia has been attributed to two mechanisms: (1) increased myocardial oxygen demand, and (2) decreased myocardial perfusion and oxygen delivery. (Willerson, J. T. et al., J. Am. Coll. Cardiol. 8(1): 245-50 (1986)). Myocardial ischemia generally appears first and is more extensive in the subendocardial region, since these deeper myocardial layers are farthest from the blood supply, with greater need for oxygen.
Transient ischemia, hibernating myocardium, and myocardial infarction are clinically different conditions.
Transient Ischemia.
The term “transient ischemia” as used herein refers to a reversible (meaning that the myocytes survive the insult) narrowing of a coronary artery at rest or with exercise where there is no thrombus or plaque rupture but where blood supply cannot be met. Every time the heart's oxygen demand increases, an imbalance between oxygen demand and supply is created. Transient ischemia produces a cascade of events beginning with metabolic and biochemical alterations leading to impaired ventricular relaxation and diastolic dysfunction, impaired systolic function, and electrocardiographic abnormalities with ST segment alterations, followed by increased end-diastolic pressure with left ventricular dyssynchrony, hypokineses, akinesis, and dyskinesis, and lastly painful symptoms of angina. Even though ischemic myocytes experience physiological and metabolic changes within seconds of the cessation of coronary flow, resulting in T wave and sometimes ST segment abnormalities (but without serum enzyme elevation), no cell death results from the ischemia. Kloner, R. A. and Jennings, R B, Circulation 104: 2981-89 (2001). Once blood flow is re-established, a complete recovery of myocyte contractile function takes place.
Although angina pectoris (chest pain) may be a symptom of transient ischemia, by and large transient ischemia is silent (meaning ST-segment depression of at least 1 mm is present without associated symptoms, e.g., chest pain) in 79% of subjects. In most patients with stable angina, for example, physical effort or emotion, with a resultant increase in heart rate, blood pressure, or contractile state, or any combination thereof, increases myocardial oxygen demand without an adequate delivery in oxygen delivery through tightly narrowed (stenosed) coronary arteries. More than 40% of patients with stable angina treated with one or more antianginal drugs have frequent episodes of silent ischemia, which has been shown to predict a higher risk of coronary events and cardiac death. Deedwania, P C, Carbajal, E V, Arch. Intern. Med. 150: 2373-2382 (1991).
Chronic Myocardial Ischemia.
The term “chronic myocardial ischemia (CMI)” as used herein refers to a prolonged subacute or chronic state of myocardial ischemia due to narrowing of a coronary blood vessel in which the myocardium “hibernates”, meaning that the myocardium downregulates or reduces its contractility, and hence its myocardial oxygen demand, to match reduced perfusion, thereby preserving cellular viability and preventing myocardial necrosis. This hibernating myocardium is capable of returning to normal or near-normal function on restoration of an adequate blood supply. Once coronary blood flow has been restored to normal or near normal and ischemia is resolved, however, the hibernating myocardium still does not contract. This flow-function mismatch resulting in a slow return of cardiac function after resolution of ischemia has been called stunning. The length of time for function to return is quite variable, ranging from days to months, and is dependent on a number of parameters, including the duration of the original ischemic insult, the severity of ischemia during the original insult, and the adequacy of the return of the arterial flow. A number of studies have provided evidence for inflammation in hibernating myocardium. Heusch, G. et al., Am. J. Physiol. Heart Circ. Physiol. 288: 984-99 (2005). A study conducted in a porcine model of myocardial hibernation in which the mean rest (left anterior descending coronary artery (LAD) coronary blood flow was reduced to about 60% of baseline for a period of 24 hours to four weeks, detected apoptotic myocytes in all experimental pigs in the hibernating regions supplied by the stenotic LAD, suggesting that functional downregulation may not be adequate to prevent gradual, ongoing myocyte death through apoptosis in hibernating myocardium. Chen, C, et al., J. Am. Coll. Cardiol. 30: 1407-12 (1997).
Acute Myocardial Infarction (AMI).
Another type of insult occurs during AML AMI is an abrupt change in the lumen of a coronary blood vessel which results in ischemic infarction, meaning that it continues until heart muscle dies. On gross inspection, myocardial infarction can be divided into two major types: transmural infarcts, in which the myocardial necrosis involves the full or nearly full thickness of the ventricular wall, and subendocardial (nontransmural) infarcts, in which the myocardial necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium. There often is total occlusion of the vessel with ST segment elevation because of thrombus formation within the lumen as a result of plaque rupture. The prolonged ischemic insult results in apoptotic and necrotic cardiomyocyte cell death. See Kajstura, J., et al., Lab Invest. 74: 86-107 (1996). Necrosis compromises the integrity of the sarcolemmal membrane and intracellular macromolecules such that serum cardiac markers, such as cardiac-specific troponins and enzymes, such as serum creatine kinase (CK), are released. In addition, the patient may have electrocardiogram (ECG) changes because of full thickness damage to the muscle. An ST-Elevation Myocardial Infarction (STEMI) is a larger injury than a non-ST-elevation myocardial infarction. ST-segment elevation and Q waves on the ECG, two features highly indicative of myocardial infarction, are seen in only about half of myocardial infarction cases on presentation.
AMI remains common with a reported annual incidence of 1.1 million cases in the United States alone (Antman, E. M., Braunwald, E., Acute Myocardial Infarction, in Principles of Internal Medicine, 15th Ed., Braunwald, E. et al., Eds., New York: McGraw-Hill (2001)). Preclinical and clinical data demonstrate that following a myocardial infarction, the acute loss of myocardial muscle cells and the accompanying peri-infarct border zone hypo-perfusion result in a cascade of events causing an immediate diminution of cardiac function, with the potential for long term persistence. The extent of myocardial cell loss is dependent on the duration of coronary artery occlusion, existing collateral coronary circulation and the condition of the cardiac microvasculature. Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990); Sheilban, I. e. al., J. Am. Coll. Cardiol. 38: 464-71 (2001); Braunwald E., Bristow, M. R., Circulation 102: IV-14-23 (2000); Rich et al., Am. J. Med. 92:7-13 (1992); Ren et al., J. Histochem. Cytochem. 49: 71-79 (2002); Hirai, T. et al., Circulation 79: 791-96 (1989); Ejiri, M. et al., J. Cardiology 20: 31-37 (1990). Because myocardial cells have virtually no ability to regenerate, myocardial infarction leads to permanent cardiac dysfunction due to contractile-muscle cell loss and replacement with nonfunctioning fibrotic scarring. Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002). Moreover, compensatory hypertrophy of viable cardiac muscle leads to microvascular insufficiency that results in further demise in cardiac function by causing myocardial muscle hibernation and apoptosis of hypertrophied myocytes in the peri-infarct border zone.
Among survivors of myocardial infarction, residual cardiac function is influenced by the extent of ventricular remodeling (meaning changes in size, shape, and function, typically a progressive decline in function, of the heart after injury). Alterations in ventricular topography (meaning the shape, configuration, or morphology of a ventricle) occur in both infarcted and healthy cardiac tissue after myocardial infarction. Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990). Ventricular dilatation (meaning a stretching, enlarging or spreading out of the ventricle) causes a decrease in global cardiac function and is affected by the infarct size, infarct healing and ventricular wall stresses. Recent efforts to minimize remodeling have been successful by limiting infarct size through rapid reperfusion (meaning restoration of blood flow) using thromobolytic agents, and mechanical interventions, including, but not limited to, placement of a stent, along with reducing ventricular wall stresses by judicious use of pre-load therapies and proper after-load management. Id. Regardless of these interventions, a substantial percentage of patients experience clinically relevant and long-term cardiac dysfunction after myocardial infarction. Sheiban, I. et al., J. Am. Coll. Cardiol. 38: 464-71 (2001). Despite revascularization of the infarct related artery circulation and appropriate medical management to minimize ventricular wall stresses, a significant percentage of these patients experience ventricular remodeling, permanent cardiac dysfunction, and consequently remain at an increased lifetime risk of experiencing adverse cardiac events, including death. Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990).
At the cellular level, immediately following a myocardial infarction, transient generalized cardiac dysfunction uniformly occurs. In the setting of a brief (i.e., lasting three minutes to five minutes) coronary artery occlusion, energy metabolism is impaired, leading to demonstrable cardiac muscle dysfunction that can persist for up to 48 hours despite immediate reperfusion. This so-called “stunned myocardium phenomenon” occurs subsequent to or after reperfusion and is thought to be a result of reactive oxygen species. The process is transient and is not associated with an inflammatory response. Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002). After successful revascularization, significant recovery from stunning occurs within three to four days, although complete recovery may take much longer. Soli, R., Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata, K. et al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al., Lancet 364: 141-48 (2004).
Coronary artery occlusion of more significant duration, i.e., lasting more than five minutes, leads to myocardial ischemia (i.e. an insufficient blood flow to the heart's muscle mass) and is associated with a significant inflammatory response that begins immediately after reperfusion and can last for up to several weeks. Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002); Frangogiannis, N. G. et al., Circulation 98: 687-798 (1998).
The inflammatory process following reperfusion is complex. Initially it contributes to myocardial damage but later leads to healing and scar formation. This complex process appears to occur in two phases. In the first so-called “hot” phase (within the first five days), reactive oxygen species (in the ischemic myocardial tissue) and complement activation generate a signal chemotactic for leukocytes (chemotaxis is the directed motion of a motile cell, organism or part towards environmental conditions it deems attractive and/or away from surroundings it finds repellent) and initiate a cytokine cascade. Lefer, D. J., Granger, D. N., Am. J. Med. 4:315-23 (2000); Frangogiannis, N. G., et al., Circulation 7:699-710 (1998). Mast cell degranulation, tumor necrosis factor alpha (TNF.alpha.) release, and increased interleukin-6 (IL-6), intercellular adhesion molecule 1 (“ICAM-1” or CD-54, a receptor typically expressed on endothelial cells and cells of the immune system), selectin (L, E and P) and integrin (CD11a, CD11b and CD18) expression all appear to contribute to neutrophil accumulation and degranulation in ischemic myocardium. Frangogiannis, N. G. et al., Circulation 7: 699-710 (1998), Kurrelmeyer, K. M, et al., Proc. Natl. Acad. Sci. USA. 10: 5456-61 (2000); Lasky, L. A., Science 258: 964-69 (1992); Ma, X. L., et al., Circulation 88(2): 649-58 (1993); Simpson, P. J. et al., J. Clin. Invest. 2: 624-29 (1998). Neutrophils contribute significantly to myocardial cell damage and death through microvascular obstruction and activation of neutrophil respiratory burst pathways after ligand-specific adhesion to cardiac myocytes. Entman, M. L., et al., J. Clin. Invest. 4: 1335-45 (1992). During the “hot” phase, angiogenesis is inhibited due to the release of angiostatic substances, including interferon gamma-inducible protein (IP 10). Frangogiannis, N. G., et al., FASEB J. 15: 1428-30 (2001).
In the second phase, the cardiac repair process begins (about day 6 to about day 14), which eventually leads to scar formation (about day 14 to about day 21) and subsequent ventricular remodeling (about day 21 to about day 90). Soon after reperfusion, monocytes infiltrate the infarcted myocardium. Attracted by complement (C5a), transforming growth factor B1 (“TGF-B1”) and monocyte chemotactic protein 1 (“MCP-1”), monocytes differentiate into macrophages that initiate the healing process by scavenging dead tissue, regulating extracellular matrix metabolism, and inducing fibroblast proliferation. Birdshall, H. H., et al., Circulation 3: 684-92 (1997). Secretion of interleukin 10 (IL-10) by infiltrating lymphocytes also promotes healing by down-regulating inflammatory cytokines and influencing tissue remodeling. Frangogiannis, N. G. et al., J. Immunol. 5:2798-2808 (2000). Mast cells also appear to be involved in the later stages of myocardial repair by participating in the formation of fibrotic scar tissue. Stem Cell Factor (SCF) is a potent attractor of mast cells. SCF mRNA has been shown to be up-regulated in ischemic myocardial segments in a canine model of myocardial infarction and thus may contribute to mast cell accumulation at ischemic myocardial sites. Franigogiannis, N. G. et al., Circulation 98: 687-798 (1998). Mast cell products (including TGF-B, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and gelatinases A and B) induce fibroblast proliferation, influence extracellular matrix metabolism, and induce angiogenesis. Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S., et al., Cardiology 93: 168-74 (2000).
Following a myocardial infarction, neoangiogenesis occurs after the “hot” phase of the inflammatory process subsides (about day 5) coincident with rising levels of VEGF (VEGF peaks at about day 7 and gradually subsides to baseline at about day 14 to about day 21). During this phase of the healing process, endothelial precursor cells (EPCs) are mobilized and recruited to the infarct site. Shinitani, S., et al., Circulation 103: 2776-79 (2001). Without being limited by theory, it has been suggested that the chemokine stromal cell derived factor-1 (SDF-1), which is the ligand for the CXCR-4 chemokine receptor expressed by CD34+ cells, also plays a role in homing of cells to areas of ischemic damage. Ceredini, D. J., et al., Nature Medicine 10: 858-63 (2004); Askari, A., et al., Lancet 362: 697-703 (2003); Yamaguchi, J. et al., Circulation 107: 1322-34 (2003). While it is known that SDF-1 plays a role in hematopoiesis and is involved in migration, homing and survival of hematopoietic progenitors, and while SDF-1 has been implicated in ischemic neovascularization in vivo by augmenting EPC recruitment to ischemic sites (Yamaguchi et al. Circulation 107:1322-1328 (2003), SDF-1's role in neoangiogenesis is not certain. There is suggestive evidence implicating SDF-1. For example, SDF-1 gene expression is upregulated during hypoxia, a deficiency of oxygen in the tissues, by hypoxia inducible factor-1. Furthermore, CD34+ cells are capable of homing to areas of ischemia, rich in SDF-1, including infarcted myocardium. Askari et al., Lancet 362: 697-703 (2003). Moreover, virtually all CD34+ CXCR-4+ cells co-express VEGF-2 and therefore migrate in response to VEGF as well as SDF-1. Peichev M., et al., Blood 95: 952-58 (2000). CD34+CXCR-4+VEGF-1 cells, once recruited, are capable of contributing to neoangiogenesis. Yamaguchi, J. et al., Circulation 107: 1322-34 (2003).
The Peri-Infarct Border Zone
The zone of dysfunctional myocardium produced by coronary artery occlusion extends beyond the infarct region to include a variable boundary of adjacent normal appearing tissue. (Hu, Q., et al., Am. J. Physiol. Heart Circ. Physiol. 291: H648-657 (2006)). This ischemic, but viable, perinfarct zone of tissue separates the central zone of progressive necrosis from surrounding normal myocardium. The peri-infarct zone does not correlate with enzymatic parameters of infarct size and is substantially larger in small infarcts. Stork, A., et al., European Radiol. 16(10): 2350-57 (2006).
Ischemia due to edema and compression of the blood vessels in the border zone may be very important to outcome after an AMI. It is known, for example, that after an AMI, transient ischemia occurs in the border zones, and that percutaneous coronary interventions, which open up the infarct-related artery, can adversely affect the health of the peri-infarct border zones. It has been suggested that intermediate levels of mean blood flow can exist as the result of admixture of peninsulas of ischemic tissue intermingled with regions of normally perfused myocardium at the border of an infarct. (Hu, Q., et al., Am. J. Physiol. Heart Circ Physiol. 291: H648-657 (2006)). However, the boundary of the intermingled coronary microvessels, which in dogs is no more than 3 mm in width, cannot explain the relatively broad region of dysfunctional myocardium surrounding an infarct. Murdock, R H, Jr., et al., Cir. Res. 52: 451-59 (1983); Buda, A J, et al., J. Am. Coll. Cardiol. 8: 150-58 (1986). Progressive dysfunction of this peri-infarct myocardium over time may contribute to the transition from compensated remodeling to progressive heart failure after an AMI.
Heart Failure
Heart failure is a complex clinical syndrome that arises secondary to abnormalities of cardiac structure and/or function that impair the ability of the left ventricle to fill or eject blood. See Hunt, S. J. Am. Coll. Cardiol. 46: e1-e82 (2005). It is a progressive condition where the heart muscle weakens and cannot pump blood efficiently. Patients may be categorized as having heart failure with depressed ejection fraction (“EF”) (referred to as “systolic failure”), or having heart failure with a normal EF or heart failure with a preserved EF (referred to as “diastolic failure”). Patients may have significant abnormalities of left ventricle (LV) contraction and relaxation and yet have no symptoms, in which case they are referred to as having “asymptomatic heart failure”. When a patient with chronic heart failure deteriorates, the patient is referred to as having “decompensated heart failure”, or, if the symptoms arise abruptly, as having “acute decompensated heart failure”.
The various diagnostic criteria used to determine the presence of heart failure are shown in the following Table (V. L. Roger, Intl. J. Environ. Res. Public Health 7(4): 1807-30 (2010)):
European SocietyGothenburg Score4Framingham1Boston2of Cardiology3 nCriteria/method of assessmentMAJORCATEGORY I:1. Symptoms ofCARDIAC SCORECRITERIA:Historyheart failure (at restHistory of heartSelf-ParoxysmalRest dyspneaor during exercise)disease (1-2 pts)reportnocturnal dyspnea(4 pts)andAngina (1-2 pts)Self-or orthopneaOrthopnea (4 pts)2. ObjectivereportNeck veinParoxysmalevidence of cardiacEdema (1 pt)Self-distensionnocturnaldysfunction (at rest)reportRalesdyspnea (3 pts)andNocturnal DyspneaSelf-CardiomegalyDyspnea on3. Response to(1pt)reportAcute pulmonarywalking on leveltreatment directedRales (1 pt)Physicaledema S3 gallop(2 pts)towards heart failureexamIncreased venousDyspnea on(in cases whereAtrial fibrillationECGpressure ≧16 cmclimbing (1 pt)diagnosis is in(1 pt)waterCATEGORYdoubt).PULMONARY SCORECirc.time ≧25 secII: PhysicalCriteria 1 and 2History of ChronicSelf-Hepatojugularexaminationshould be fulfilled inbronchitis/asthmareportrefluxHeart rateall cases(1-2 pts)MINORabnormality (1-2Cough, phlegm, orSelf-CRITERIA:pts)wheezing (1 pt)reportAnkle edemaJugular venousRhonchi (2 pts)PhysicalNight coughpressureexamDyspnea onelevation (1-2Cardiac and pulmonary score areexertionpts)calculated and used toHepatomegalyLung cracklesdifferentiate Cardiac formPleural effusion(1-2 pts)pulmonary dyspneaVital capacityWheezing (3 pts)decreased 1/3Third heartfrom maximumsound (3 pts)Tachycardia rateCATEGORYof ≧120/min)III: ChestMAJOR ORradiographyMINORAlveolarCRITERION:pulmonaryWeight loss ≧4.5 kgedema (4 pts)in 5 days inInterstitialresponse topulmonarytreatmentedema (3 pts)HEARTBilateral pleuralFAILURE:effusions (3 pts)present with 2Cardiothoracicmajor or 1 majorratio ≧0.50 (3and 2 minorpts)criteriaUpper-zone flowredistribution (2pts)HEARTFAILURE:Definite 8-12pts, possible 5-7pts, unlikely 4pts or less1McKee PA, Castelli WP, McNamara PM, Kannel WB. The natural history of congestive heart failure: the Framingham study. N. Engl. J. Med. 285: 1441-1446 (1971)2Carlson K J, Lee DC Goroll AH, Lehy M, Johnson RA, an analysis of physicians' reasons for prescribing long-term digitalis therapy in outpatients. J. Chronic Dis. 38: 733-39 (1985)3Guidelines for the diagnosis of heart failure The Task Force on Heart Failure of the European Society of Cardiology. Eur. Heart J. 16: 741-751 (1995)4Eriksson H, Caidahl K, Larsson B, Ohlson LO, Welin L, Wilhelmsen L, Svardsudd K. Cardiac and pulmonary causes of dyspnoea-validation of a scoring test for clinical-epidemiological use: the Study of Men Born in 1913. Eur. Heart J. 8: 1007-1014 (1987)
The prognosis of heart failure is poor with reported survival estimates of 50% at 5 years and 10% at 10 years; left ventricular dysfunction is associated with an increase in the risk of sudden death. Id.
To date, no ideal therapy exists for preventing the long term adverse consequences of vascular insufficiency, particularly vascular insufficiency after myocardial infarction. Large vessel revascularization (meaning the successful placement of a stent) is insufficient in addressing increased demands posed by compensatory myocardial hypertrophy. As a result, infarct extension and fibrous replacement commonly occur, regardless of large vessel revascularization, appropriate medical management of ventricular wall stresses, and potential natural, albeit suboptimal, CD34+ cell-mediated neoangiogenesis (one of theories relating to the underlying cause of myocardial infarction is that the ability to mobilize these cells may be biologically limited).
Intense interest has developed in evaluating the ability of endothelial and myocardial precursor cells to limit damage to the myocardium after infarction and to limit or prevent ventricular remodeling. Significant preclinical data and some clinical data demonstrate the safety and potential of cell therapy using a variety of cell precursors (particularly hematopoietic cells) to contribute to neoangiogenesis, limited cardiac myogenesis (principally by fusion), and muscle preservation in the myocardial infarct zone. See, e.g., Jackson, et al., J. Clin. Invest. 107: 1395-1402 (2001); Edelberg, J. M., et al., Cir. Res. 90: e89-e93 (2002); Schichinger, V. et al., New Engl. J. Med. 355 (12): 1210-21 (2006) (using bone marrow-derived progenitor cells); Assmus, B. et al., New Engl. J. Med. 355 (12) 1222-32 (2006) (using bone marrow-derived progenitor cells), but see Lunde, K. et al., New Eng. J. Med. 355 (12): 1199-209 (2006) (using hone marrow-derived progenitor cells).
Bone marrow consists of a variety of precursor and mature cell types, including hematopoietic cells (the precursors of mature blood cells) and stromal cells (the precursors of a broad spectrum of connective tissue cells), both of which appear to be capable of differentiating into other cell types. Wang, J. S. et al., J. Thorn. Cardiovasc. Surg. 122: 699-705 (2001); Tomita, S. et al., Circulation 100 (Suppl. II): 247-256 (1999); Saito, T. et al., Tissue Eng. 1: 327-43 (1995). Unmodified (i.e., not fractionated) marrow or blood-derived cells have been used in several clinical studies, for example, Hamann, K. et al., Japan Cir. J. 65: 845-47 (2001); Strauer, B. E., et al., Circulation 106: 1913-18 (2002); Assmus, et al., Circulation 106: 3009-3017 (2002); Dobert, N. et al., Eur. J. Nuel. Med. Mal. Imaging, 8: 1146-51 (2004); Wollert, K. C. et al., Lancet 364: 141-48 (2004). Since the mononuclear fraction of bone marrow contains stromal cells, hematopoietic precursors, and endothelial precursors, the relative contribution of each of these populations to the observed effects, if any, remains unknown.
CD34 is a hematopoietic stem cell antigen selectively expressed on hematopoietic stem and progenitor cells derived from human bone marrow, blood and fetal liver. Yin et al., Blood 90: 5002-5012 (1997); Miaglia, S. et al., Blood 90: 5013-21 (1997). Cells that express CD34 are termed CD34+. Stromal cells do not express CD34 and are therefore termed CD34−. CD34+ cells isolated from human blood may be capable of differentiating into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. See Yeh, et al., Circulation 108: 2070-73 (2003). CD34+ cells represent approximately 1% of bone marrow derived nucleated cells; CD34 antigen also is expressed by immature endothelial cell precursors (mature endothelial cells do not express CD34). Peichev, M. et al., Blood 95: 952-58 (2000). In vitro, CD34+ cells derived from adult bone marrow give rise to a majority of the granulocyte/macrophage progenitor cells (CFU-GM), some colony-forming units-mixed (CFU-Mix) and a minor population of primitive erythroid progenitor cells (burst forming units, erythrocytes or BFU-E). Yeh, et al., Circulation 108: 2070-73 (2003). CD34+ cells also may have the potential to differentiate into, or to contribute to, the development of new myocardial muscle, albeit at low frequency.
Techniques have been developed using immunomagnetic bead separation to isolate a highly purified and viable population of CD34+ cells from bone narrow mononuclear cells. See U.S. Pat. Nos. 5,536,475, 5,035,994, 5,130,144, 4,965,205, the contents of each of which is incorporated herein by reference. Two clinical studies support the clinical application of bone marrow derived CD34+ cells after myocardial infarction. See C. Stamm, et al., Lancet 361: 45-46 (2003); Herenstein, B. et al., Blood Supplement, Abs. 2696 (2004).
Neoangiogenesis
Neovascularization, the formation of new blood vessels, is inherent in vascular tissue, and it can be induced by trauma, ischemia, inflammation, or tumor growth. The creation of new blood vessels is dependent on a complicated interaction between locally produced cytokines and cells derived from the tissue area, and blood circulation. Neovascularization can be divided into three processes: angiogenesis, vasculogenesis, and arteriogenesis.
Angiogenesis is the formation of new capillaries by sprouting from the existing capillary net, probably from the postcapillary venules; arteriogenesis is the transformation of preexisting aterioles/collaterales into small muscular arteries and/or de novo formation of new vessels with a tunica media; and vasculogenesis is the formation of new vessels from multipotent endothelial stem cells.
In vasculogenesis, circulating endothelial progenitor cells (EPC) contribute to new blood vessel growth (capillaries) by secreting the necessary growth factors and chemokines for endothelial cells to migrate or by incorporating into the newly formed vessels. During angiogenesis, endothelial cells are activated by ischemia and grow in the direction of angiogenic signals. The endothelial cells fuse and develop a lumen, thereby forming a new, small capillary vessel.
In arteriogenesis, circulating leukocytes are attracted to the activated endothelium. They assist in enlarging collateral anastomosis (connection of two blood vessels).
Angiogenic Growth Factors
Neoangiogenesis (meaning formation of new or recent blood vessels), is dependent on a complex interaction between extracellular matrix, endothelial cells and pericytes in response to an imbalance in the presence of angiogenic, as compared to angiostatic factors in the local environment (Fangogiannis, N G, The FASEB Journal, Vol. 15, June 2001, pp. 1428-1430).
A large number of angiogenic factors and their receptors have been identified including basic fibroblast growth factor, platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor, fibroblast growth factor, angiopoietin-1, transforming growth factor beta-1 (TGF-β1), transforming growth factor alpha (TGF-α), and epidermal growth factor (EGF) (Rifkin D B, Moscatelli D., J Cell Biol 1989; 109:1-6; Nicosia R F, Nicosia S V, Smith M., Am J Pathol 1994; 145:1023-1029; Takahashi Y, Bucana C D, Liu W et al. J Natl Cancer Inst 1996; 88:1146-1151; Jouanneau J, Moens G, Montesano R et al., Growth Factors 1995; 12:37-47; Suri C, McClain J, Thurston G et al. Science 1998; 282:468-471; Pepper M S, Vassalli J D, Orci L et al., Exp Cell Res 1993; 204:356-363; Gleave M E, Hsieh J T, Wu H C et al., Cancer Res 1993; 53:5300-5307).
Angiogenesis in the tissue can be initiated by local production and liberation of vascular growth factors. Many different vascular growth factors have now been discovered, which can induce angiogenesis by stimulation of growth and migration of endothelial cells. Angiogenic potentials of growth factors depend on their ability to induce a proliferation of endothelial cells, a modification in cell adhesion (i.e., cell to cell and cell to extracellular matrix peptides interactions) and/or a remodeling process of the extracellular matrix and the basement membrane. Main growth factors are direct-acting endothelial cell mitogens (e.g., FGF, VEGF, HGF). Other factors (PDGF, TGF-beta, and TNF-alpha) can be indirectly mitogenic, by promoting an overexpression of a direct-acting mitogen. Members of the CXC chemokine family also have been reported to play a role in the regulation of angiogenesis. CXC chemokines behave as either angiogenic or angiostatic, depending on the presence of an ELR protein motif (ELR positive chemokines such as IL-8 are angiogenic, ELR negative chemokines such as IP-10 are angiostatic).
During recent years, a number of experimental studies have shown that treatment with angiogenic growth factors can promote the development of collaterals to ischemic tissue in models of progressive coronary occlusion, and acute myocardial infarction.
Direct-Acting Endothelial Cell Mitogens
Vascular Endothelial Growth Factor (VEGF)
VEGF is a diffusible endothelial cell-specific mitogen and angiogenic factor that also increases vascular permeability (Ferrara N, Davis-Smyth T., Endocr Rev 1997; 18:4-25; Torimura T, Sata M, Ueno T et al. Hum Pathol 1998; 29:986-991). It elicits a pronounced angiogenic response in a variety of in vivo models (Connolly D T, Heuvelman D M, Nelson R et al., J Clin Invest 1989; 84:1470-1478; Plate K H, Breier G, Weich H A et al. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992; 359:845-848; Phillips G D, Stone A M, Jones B D et al., In Vivo 1994; 8:961-965; Tolentino M J, Miller J W, Gragoudas E S et al. Arch Ophthalmol 1996; 114:964-970; Cao Y, Linden P, Farnebo J et al. Proc Natl Acad Sci USA 1998; 95:14389-14394). Endothelial cell survival in newly formed vessels is VEGF-dependent (Alon T, Hemo I, Itin A et al. Nat Med 1995; 1:1024-1028). Three high-affinity cognate endothelial receptors for VEGF have been identified: VEGFR-1/Flt-1, VEGFR-2/Flk-1/KDR, and VEGFR-3/Flt-4. These receptors function as signaling molecules during vascular development (Mustonen T, Alitalo K., J Cell Biol 1995; 129:895-898). VEGFR-1 and VEGFR-2 are cell surface receptor tyrosine kinases (RTKs), which are localized on endothelial cells during embryogenic development. VEGF RTKs, members of a large family of RTKs, are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration, and metabolism. VEGFR-2 is exclusively expressed in endothelial cells and appears to play a pivotal role in endothelial cell differentiation and vasculogenesis (Millauer B, Wizigmann-Voos S, Schnurch H et al., Cell 1993; 72:835-846; Quinn T P, Peters K G, De Vries C et al., Proc Natl Acad Sci USA 1993; 90:7533-7537).
Hypoxia can directly increase the synthesis of VEGF receptors, permitting a tissue-targeting of VEGF during ischemia induced angiogenesis. Beyond angiogenesis, the permanent expression of VEGF receptors by quiescent endothelium suggests a role of VEGF in the maintenance of endothelial integrity. Recently, among CD34+ mononuclear blood cells, some putative circulating endothelial cell progenitors have been isolated, which are incorporated in post-natal angiogenic sites. Flk-1 is expressed by those monocytes, assimilated to angioblasts, and by early hematopoietic stem cells, but ceases to be expressed as soon as hematopoietic differentiation is initiated towards non-endothelial specificities. Flt-1, also present on monocytes seems to be involved in monocyte chemotaxis. Bautch, V L, Blood 2006 107: 3-4, DOI: 10.1182/blood-2005-10-4061; Feistritzer, C et al., Am. J. Respir. Cell Mol. Biol., Vol. 30, pp. 729-735, 2004; Itokawa, T et al., Mol Cancer Ther 2002; 1: 295-302; Patschan, D et al., Am J Physiol Renal Physiol 291: F176-F185, 2006.
In vitro, VEGF stimulates the proliferation and migration of endothelial cells and forms tube-like structures, but it can also inhibit endothelial cell apoptosis. Upregulation of plasminogen activators and collagenases by VEGF induces extracellular matrix proteolysis. This allows the migration of endothelial cells and release of growth factors responsible for an auto-amplification. In addition, VEGF augments vessels permeability and may cause extravasation of plasma proteins necessary for the formation of a new extracellular matrix. It can also accelerate reendothelialization, attenuate intimal hyperplasia in balloon-injured carotid artery. Wang, S et al., PNAS, Vol. 105, No. 22, pp. 7738-7743; Bernatchez, P N et al., The Journal of Biological Chemistry, Vol. 274, No. 43, Oct. 22, 1999, pp. 31047-31054; Asahara, T et al., Circulation, 1995; 91: 2793-2801.
Fibroblast Growth Factor (FGF)
FGF comprises sixteen members (FGF-1 to FGF-16) with a wide range of targets and potentials. Two of them have been studied concerning their angiogenic power: acidic FGF (or FGF-1) and basic-FGF (or FGF-2). Four isoforms of FGF-2 (18 to 24 kD) have been described, and have widespread expression. Binding of FGF-1 or FGF-2 to heparan sulphates enhances their autocrine or paracrine bioactivity.
FGF-receptors originate from the super-family of immunoglobulins, with 4 types identified: FGF-R1, R2, R3, and R. With variable affinity, those tyrosine kinase-type receptors have been isolated on cardiomyocytes, endothelial and smooth muscle cells. In vitro, stimulation of endothelial cells by FGF-2 leads to the formation of capillary-like tubular structures, subsequent to proliferation and migration of endothelial cells.
The physiological involvement of FGF during ischemia-induced angiogenesis is not entirely understood.
Scatter Factor/Hepatocyte Growth Factor (SH/HGF)
HGF, a 80 kDa heterodimeric cytokine, binds heparan sulfate as soon as it is secreted by mesenchymal cells. Ischemia was found to be a positive stimulus of expression of both HGF and its tyrosine kinase receptor. In vitro, the formation of tube-like structures, subsequent to HGF's direct activation of endothelial cell proliferation and migration, is highly enhanced by upregulation of VEGF-A in smooth muscle cells.
Other Mitogens
Angiopoietin-1 (Ang-1)
Angiopoietin-1, expressed by mesenchymal cells, is considered to be the principal ligand of TIE-2, a tyrosine kinase receptor, which is expressed on vascular endothelial cells and early hematopoietic cells. Angiopoietin-2 (ang-2) is a second ligand that inhibits TIE-2 phosphorylation, and acts like a competitive inhibitor of Ang-1.
Platelet Derived Growth Factor (PDGF)
Platelets, macrophages, endothelial cells and vascular smooth muscle cells provide three kinds of PDGF: AA, BB, or AB. Shear-stress and hypoxia have been reported to upregulate PDGF-BB expression in endothelial cells and in macrophages. Shear-stress activates PDGF-β receptors expressed by endothelial cells, smooth muscle cells and pericytes.
PDGF is considered to be a proangiogenic factor. It is not a direct mitogen for endothelial cells, but is able to enhance production of both FGF-2 and VEGF by smooth muscle cells. Recruitment of smooth muscle cells and pericytes represents probably the principal contribution of PDGF during angiogenesis,
Transforming Growth Factor (TGF)
TGF-β is a 25 kDa homodimeric polypeptide; its distribution includes kidneys, liver, heart, platelets, and endothelial cells. TGF-β production is sensitive to the variation of shear stress exerted on endothelial cells. TGF-β is considered to be a positive regulator of angiogenesis in vivo.
Apoptotic Pathways
Apoptotic cell death is induced by many different factors and involves numerous signaling pathways, some dependent on caspase proteases (a class of cysteine proteases) and others that are caspase independent. It can be triggered by many different cellular stimuli, including cell surface receptors, mitochondrial response to stress, and cytotoxic T cells, resulting in activation of apoptotic signaling pathways.
The caspases involved in apoptosis convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases that then degrade other cellular targets that lead to cell death. The caspases at the upper end of the cascade include caspase-8 and caspase-9. Caspase-8 is the initial caspase involved in response to receptors with a death domain (DD) like Fas.
Receptors in the TNF receptor family are associated with the induction of apoptosis, as well as inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. The Fas-FasL interaction plays an important role in the immune system and lack of this system leads to autoimmunity, indicating that Fas-mediated apoptosis removes self-reactive lymphocytes. Fas signaling also is involved in immune surveillance to remove transformed cells and virus infected cells. Binding of Fas to oligimerized FasL on another cell activates apoptotic signaling through a cytoplasmic domain termed the death domain (DD) that interacts with signaling adaptors including FAF, FADD and DAX to activate the caspase proteolytic cascade. Caspase-8 and caspase-10 first are activated to then cleave and activate downstream caspases and a variety of cellular substrates that lead to cell death.
Mitochondria participate in apoptotic signaling pathways through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals, and activates Apaf-1, a protease released from mitochondria. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac/DIABLO is released from mitochondria and inhibits IAP proteins that normally interact with caspase-9 to inhibit apoptosis. Apoptosis regulation by Bcl-2 family proteins occurs as family members form complexes that enter the mitochondrial membrane, regulating the release of cytochrome c and other proteins. TNF family receptors that cause apoptosis directly activate the caspase cascade, but can also activate Bid, a Bcl-2 family member, which activates mitochondria-mediated apoptosis. Bax, another Bcl-2 family member, is activated by this pathway to localize to the mitochondrial membrane and increase its permeability, releasing cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation, blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria that is released from mitochondria by apoptotic stimuli. While cytochrome C is linked to caspase-dependent apoptotic signaling, AIF release stimulates caspase-independent apoptosis, moving into the nucleus where it binds DNA. DNA binding by AIF stimulates chromatin condensation, and DNA fragmentation, perhaps through recruitment of nucleases.
The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, causing self-cleavage and activation of caspase-9. Caspase-3, -6 and -7 are downstream caspases that are activated by the upstream proteases and act themselves to cleave cellular targets.
Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores, and triggering apoptosis, perhaps through cleavage of caspases, although caspase-independent mechanisms of Granzyme B mediated apoptosis have been suggested.
Fragmentation of the nuclear genome by multiple nucleases activated by apoptotic signaling pathways to create a nucleosomal ladder is a cellular response characteristic of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse (CAD). DFF/CAD is activated through cleavage of its associated inhibitor ICAD by caspases proteases during apoptosis. DFF/CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structure and perhaps recruit CAD to chromatin. Another apoptosis activated protease is endonuclease G (EndoG). EndoG is encoded in the nuclear genome but is localized to mitochondria in normal cells. EndoG may play a role in the replication of the mitochondrial genome, as well as in apoptosis. Apoptotic signaling causes the release of EndoG from mitochondria. The EndoG and DFF/CAD pathways are independent since the EndoG pathway still occurs in cells lacking DFF.
Hypoxia, as well as hypoxia followed by reoxygenation can trigger cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or potentiate apoptosis due to many stimuli that activate the mitochondrial cell death pathway. Loberg, R D, et al., J. Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated to induce caspase 3 activation and to activate the proapoptotic tumor suppressor gene p53. It also has been suggested that GSK-3 promotes activation and translocation of the proapoptotic Bcl-2 family member, Bax, which, upon aggregation and mitochondrial localization, induces cytochrome c release. Akt is a critical regulator of GSK-3, and phosphorylation and inactivation of GSK-3 may mediate some of the antiapoptotic effects of Akt.
Akt (also known as protein kinase B) is a 60 kDa serine/threonine kinase. It is activated in response to stimulation of tyrosine kinase receptors such as platelet-derived growth factor (PDGF), insulin-like growth factor, and nerve growth factor (Shimamura, H, et al., J. Am. Soc. Nephrol. 14: 1427-1434, 2003; Datta K, Franke T F, Chan T O, Makris A, Yang S I, Kaplan D R, Morrison D K, Golemis E A, Tsichlis P N, Mol Cell Biol 15: 2304-2310, 1995; Kulik G, Klippel A, Weber M J, Mol Cell Biol 17: 1595-1606, 1997; Yao R, Cooper G M, Science 267: 2003-2006, 1995). Stimulation of Akt has been shown to be dependent on phosphatidylinositol 3-kinase (PI3-kinase) activity (Fruman D A, Meyers R E, Cantley L C, Annu Rev Biochem 67: 481-507, 1998; Choudhury G G, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, Abboud H E, Am J Physiol 273: F931-938, 1997, Franke T F, Yang S I, Chan T O, Datta K, Kazlauskas A, Morrison D K, Kaplan D R, Tsichlis P N, Cell 81: 727-736, 1995; Franke T F, Kaplan D R, Cantley L C, Cell 88: 435-437, 1997).
Akt has been shown to act as a mediator of survival signals that protect cells from apoptosis in multiple cell lines (Brunet A, Bonni A, Zigmond M J, Lin M Z, Juo P, Hu L S, Anderson M J, Arden K C, Blenis J, Greenberg M E, Cell 96: 857-868, 1999; Downward J, Curr Opin Cell Biol 10: 262-267, 1998). For example, phosphorylation of the pro-apoptotic Bad protein by Akt was found to decrease apoptosis by preventing Bad from binding to the anti-apoptotic protein Bcl-XL (Dudek H, Datta S R, Franke T F, Birnbaum M J, Yao R, Cooper G M, Segal R A, Kaplan D R, Greenberg M E, Science 275: 661-665, 1997; Datta S R, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg M E, Cell 91: 231-241, 1997). Akt was also shown to promote cell survival by activating nuclear factor-kB (NF-kB) (Cardone M H, Roy N, Stennicke H R, Salvesen G S, Franke T F, Stanbridge E, Frisch S, Reed J C, Science 282: 1318-1321, 1998; Khwaja A, Nature 401: 33-34, 1999) and inhibiting the activity of the cell death protease caspase-9 (Kennedy S G, Kandel E S, Cross T K, Hay N, Mol Cell Biol 19: 5800-5810, 1999).
Animal Models
Peripheral artery disease (PAD), also called peripheral vascular disease (PVD), is modeled by the hind limb model of ischemia in which the femoral artery of the mouse is tied off to simulate peripheral artery disease. PAD, which commonly affects the arteries supplying the leg and includes all diseases caused by the obstruction of large arteries in the arms and legs, can result from atherosclerosis, inflammatory processes leading to stenosis, an embolism or thrombus formation. Restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking (intermittent claudication). Any further reduction in blood flow causes ischemic pain at rest. This condition is called chronic limb ischemia, meaning the demand for oxygen cannot be sustained when resting. Ulceration and gangrene may then supervene in the toes, which are the furthest away from the blood supply, and can result in loss of the involved limb if not treated.
Therapies for limb ischemia have the goals of collateral development and blood supply replenishment. Bone marrow derived CD34+mononuclear cells have been tested in such hindlimb ischemia models, but the hindlimb ischemia model does not model what takes place in the heart. A preferred therapy after AMI would stop cells from dying during recovery that leads to reverse remodeling and failure, or replace the dying cells with cardiomyocytes.
The closest animal model, the pig model, is not a good model of human disease because (i) all experiments generally are done in nonatherosclerotic animals, (ii) the animals are not treated with angioplasty, (iii) normal pigs do not embolize blood vessels; (iv) circulation of the pig is not exactly the same as human; and (iv) the peri-infarct border zone may not be the same.
A marginal improvement in angina symptoms recently was reported when CD34+ cells were mobilized with G-CSF, apheresed after 5 days, and then injected into an ischemic area of the heart based on Naga mapping. [Northwestern University (2009 Apr. 1). Adult Stem Cell Injections May Reduce Pain And Improve Walking In Severe Angina Patients. ScienceDaily. Retrieved Oct. 21, 2010, from http://www.sciencedaily.com-/releases/2009/03/090330091706.htm] Data from a phase I trial conducted by the present inventors has provided evidence that subjects treated with at least 10×106 isolated autologous CD34+hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity (n=9) experienced significant improvement in resting perfusion rates at 6 months compared to subjects receiving 5 million cells (n=6) and control (n=15), as measured by the SPECT Total Severity Score (−256 versus +13, p=0.01). U.S. Patent Applications 61/169,850 and 61/119,552, incorporated herein by reference.
The described invention is a therapy for preventing the long-term adverse consequences of vascular insufficiency, particularly vascular insufficiency that produces expansion of the myocardial infarct area after an AMI progressing to heart failure. Administration of a potent dose of a nonexpanded, isolated population of autologous mononuclear cells enriched for CD34+ cells, which further contains a subpopulation of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity, administered early or late after occurrence of an AMI can result in a reduction in major adverse cardiac events, including, but not limited to, premature death, recurrent myocardial infarction, the development of congestive heart failure, significant arrhythmias, and acute coronary syndrome, and the worsening of congestive heart failure, significant arrhythmias, and acute coronary syndrome. Paracrine effects of the chemotactic hematopoietic stem cell product used in the claimed methods prevent the fragile cardiomyocytes in the peri-infarct border zone from dying, which prevents progressive myocardial cell loss, and which leads to improvement in function and a reduction of risk of major adverse cardiovascular events. In other words, 10×106 CD34+ potent cells administered in a clinical setting is used as a surrogate to provide enough CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity and that move in response to SDF-1 to effect a biological effect (paracrine and neoangiogenic), which prevents cardiomyocyte cell death and later changes consistent with ventricular modeling.